Abstract
Hepatocyte nuclear factor 4alpha (HNF4alpha) is a member of the nuclear receptor superfamily that plays a central role in organ development and metabolic functions. Mutations on HNF4alpha cause maturity-onset diabetes of the young (MODY), a dominant monogenic cause of diabetes. In order to understand the molecular mechanism of promoter recognition and the molecular basis of disease-causing mutations, the recombinant HNF4alpha DNA-binding domain was prepared and used in a study of its binding properties and in crystallization with a 21-mer DNA fragment that contains the promoter element of another MODY gene, HNF1alpha. The HNF4alpha protein displays a cooperative and specific DNA-binding activity towards its target gene-recognition elements. Crystals of the complex diffract to 2.0 A using a synchrotron-radiation source under cryogenic (100 K) conditions and belong to space group C2, with unit-cell parameters a = 121.63, b = 35.43, c = 70.99 A, beta = 119.36 degrees . A molecular-replacement solution has been obtained and structure refinement is in progress. This structure and the binding studies will provide the groundwork for detailed functional and biochemical studies of the MODY mutants.
Highlights
Hepatocyte nuclear factor 4 (HNF4 ) is a tissue-specific transcription factor that plays an essential role in early vertebrate development and embryonic survival
In order to elucidate the molecular basis of HNF4 function and the monogenic causes of diabetes, we have prepared and crystallized the human HNF4 DNA-binding domain in complex with a high-affinity HNF1 promoter element containing the HNF4 recognition sequence
Gel-filtration experiments showed that the HNF4 -DNA-binding domain (DBD) protein existed as a monomer in solution (Fig. 2)
Summary
Hepatocyte nuclear factor 4 (HNF4 ) is a tissue-specific transcription factor that plays an essential role in early vertebrate development and embryonic survival It regulates the expression of a wide variety of essential genes, including those involved in liver and pancreatic cell differentiation (Li et al, 2000; Odom et al, 2004), embryogenesis and early development (Duncan et al, 1994; Lausen et al, 2000), glucose metabolism (Stoffel & Duncan, 1997), lipid homeostasis (Hayhurst et al, 2001) and amino-acid metabolism (Schrem et al, 2002). In order to elucidate the molecular basis of HNF4 function and the monogenic causes of diabetes, we have prepared and crystallized the human HNF4 DNA-binding domain in complex with a high-affinity HNF1 promoter element containing the HNF4 recognition sequence
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